Process for the manufacture of degarelix and its intermediates

ABSTRACT

The present invention relates to a liquid (or solution)-phase manufacturing process for preparing the decapeptide Degareiix, its amino-protected precursor, and other useful intermediates. The invention further relates to polypeptides useful in the solution-phase manufacturing process and to the purification of Degareiix itself. The manufacturing process comprises the step of cleaving the ε-amino protecting group Pε from a Degareiix precursor according to formula {P 4 )(Pε)Ac-AA 1 -AA 10 -NH 2  in an organic solvent comprising the precursor and a cleaving agent dissolved therein: wherein P 4  is a hydroxyl-protecting group or hydrogen, preferably hydrogen.

TECHNICAL FIELD

The present invention relates to a liquid (or solution)-phasemanufacturing process for preparing the decapeptide Degarelix, itsamino-protected precursor, and other useful intermediates. The inventionfurther relates to polypeptides useful in the solution-phasemanufacturing process and to the purification of Degarelix itself.

BACKGROUND OF THE INVENTION

Prostate cancer is a leading cause of morbidity and mortality for men inthe industrialised world. Degarelix, also known as FE200486, is a thirdgeneration gonadotropin releasing hormone (GnRH) receptor antagonist (aGnRH blacker) that has been developed and recently approved for prostatecancer patients in need of androgen ablation therapy (Doehn et al.,Drugs 2006, vol. 9, No. 8, pp. 565-571; WO 09846634). Degarelix acts byimmediate and competitive blockade of GnRH receptors in the pituitaryand, like other GnRH antagonists, does not cause an initial stimulationof luteinizing hormone production via the hypothalamic-pituitary-gonadalaxis, and therefore does not cause testosterone surge or clinical flare(Van Poppel, Cancer Management and Research, 2010:2 39-52; Van Poppel etal., Urology, 2008, 71(6), 1001-1006); James, E. F. et al., Drugs, 2009,69(14), 1967-1976).

Degarelix is a synthetic linear decapeptide containing seven unnaturalamino acids, five of which are D-amino acids. It has ten chiral centersin the back bone of the decapeptide. The amino acid residue at position5 in the sequence has an additional chiral center in the side-chainsubstitution giving eleven chiral centers in total. Its CAS registrynumber is 214766-78-6 (of free base) and it is commercially availableunder the Trademark Firmagon™. The drug substance is chemicallydesignated as D-Alaninamide,N-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L-seryl-4-[[[(4S)-hexahydro-2,6-diaxo-4-pyrimidinyl]carbonyl]amino]-L-phenylalanyl-4-[(aminocarbonyl)amino]-D-phenylalanyl-L-leucyl-N6-(1-methylethyl)-L-lysyl-L-prolyl-and is represented by the chemical structure below:

The structure of Degarelix can also be represented as:

Ac-D-2NaI-D-4Cpa-D-3PaI-Ser-4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH₂

where Ac is acetyl, 2NaI is 2-naphthylalanine, 4 Cpa is4-chlorophenylalanine, 3 PaI is 3-pyridylalanine, Ser is serine, 4Aph is4-aminophenylalanine, Hor is hydroorotyl, Cbm is carbamoyl, Leu isleucine, Lys(iPr) is N6-isopropyllysine, Pro is proline and Ala isalanine.

For the purposes of describing this invention, each amino acid inDegarelix will be given the shorthand notation as follows:

AA₁ is D-2NaI, AA₂ is D-4 Cpa, AA₃ is D-3 PaI, AA₄ is Ser, AA₅ is4Aph(L-Hor), AA₆ is D-Aph(Cbm), AA₇ is Leu, AA₈ is Lys(iPr), AA₉ is Proand AA₁₀ is D-Ala.

Thus, as an example, Degarelix can be represented as Ac-AA₁-AA₁₀-NH₂,the tetrapeptide Ac-D-2NaI-D-4 Cpa-D-3 PaI-Ser can be represented asAc-AA₁-AA₄ and the hexapeptide4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH₂ as AA₅-AA₁₀-NH₂.

Degarelix has previously been prepared using Boc-solid phase peptidesynthesis (SPPS) methodology as reported in WO 98/46634 and Jiang etal., J. Med. Chem. 2001, 44, 453-467. Basically, Boc-protected D-Ala isfirst coupled to MBNA resin in dimethylformamide (DMF)/CH₂Cl₂ usingdiisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) asactivating or coupling agents. Once D-Ala is coupled to the resin,synthesis proceeds by washing, deblocking and then coupling the nextamino acid residue until the decapeptide has been completed. The sidechain primary amino groups of 4Aph in the 5-position and of D-4Aph inthe 6-position are protected by Fmoc when they are added and modifiedwith L-Hor and Cbm respectively before the next amino acid in the chainis added. This requires the additional steps of first removing theside-chain protection with piperidine, reacting the newly freed aminogroup on the peptidoresin with tert-butyl isocyanate or L-hydrooroticacid, ensuring that the reaction is complete with a ninhydrin test andthen washing the peptidoresin before adding the next amino acid residue(see also Sorbera et al., Drugs of the Future 2006, Vol. 31, No. 9, pp755-766).

While Boc-SPPS methodology has afforded sufficient quantities ofDegarelix until now, the growing demand for this polypeptide means thatever larger quantities are required. Boc-SPPS, which requires HFcleavage, is not suited to large scale industrial synthesis. Indeed, WO98/46634 mentions that SPPS is only suitable for limited quantities ofup to 1 kg while classical peptide solution synthesis, or liquid phasepeptide synthesis (LPPS), is preferred for larger quantities of product.WO 98/46634 does not specify how such synthesis should be performed.Further, expense attributable the large excess of coupling reagents,additives, and amino acids required for the SPPS. While the existence ofa liquid phase peptide synthesis of Degarelix has been reported [EMEAReport: Assessment Report for Firmagon™ (Degarelix): Doc. Ref.EMEA/CHMP/635761/2008], as of now no details of such a process have beenpublically disclosed.

WO 97/34923 and WO 99/26964 are international Application Publicationswhich are concerned with liquid phase processes for the preparation ofbiologically active peptides. WO 99/26964 is particularly concerned withthe liquid phase synthesis of decapeptides having activity as GnRHantagonists. WO 99/26964 lists a number of inherent limitations of theSPPS methodology for producing GnRH antagonists including the limitedcapacity of the resin, the large excess of reagents and amino acidsrequired, as well as the need to protect all reactive side chains suchas the hydroxy group in Ser, the aromatic amino groups in Aph and D-Aph,the ε-i-propylamino group in Lys(i-Pr).

International Application Publication No. WO 99/26964 describes a liquidphase process which involves first preparing the central peptidefragments of the 5 and 6 positions of a decapeptide with the side chainsfully elaborated and then assembling the peptide through a “4-2-4”,“3-3-4” or “3-4-3” fragment assembly pattern. For example, in thepreparation of the GnRH antagonist Azaline B, a tetrapeptide is coupledwith a hexapeptide to form the desired decapeptide. When the samefragment assembly pattern is attempted for Degarelix, racemisation ofthe Serine amino acid (AA₄) occurs resulting in about 20% impurity ofL-Ser. This impurity carries over into the final decapeptide and isdifficult to remove. Furthermore, when preparing the tetrapeptideAA₁-AA₄ by adding the Ser unit to the tripeptide AA₁-AA₃ following theprocedure described in WO 99/26964, tetrabutylammonium ions from thehydrolysis of the benzyl ester group could not be removed completelyduring the subsequent operations and were carried through to the finalproduct. It was further found that in the Degarelix synthesis, theL-hydroorotyl group rearranges to its hydantainacetyl analogue whenL-dihydroorotic acid is coupled with 4 Amp to prepare AA₅. These andother problems with the solution-phase synthesis of Degarelix have nowbeen overcome and a new solution-phase polypeptide synthesis of thisdecapeptide is disclosed herein for the first time.

SUMMARY OF THE INVENTION

The problems of SSPS methods for preparing Degarelix and the drawbacksof LLPS methods as described in WO 97/34923 and WO 99/26964 have nowbeen overcome and are the subject of this invention.

In general, this invention relates to a liquid-phase synthesis of thedecapeptide Degarelix.

In one aspect, the invention relates to a liquid-phase process forpreparing Degarelix having the formula Ac-AA₁-AA₁₀-NH₂ or apharmaceutically acceptable salt or solvate thereof, comprising the stepof cleaving the ε-amino protecting group Pε from a Degarelix precursoraccording to formula (P₄)(Pε)Ac-AA₁-AA₁₀-NH₂ in an organic solventcomprising the precursor and a cleaving agent dissolved therein:

P₄ is a hydroxyl-protecting group or hydrogen, preferably, tBu, (ψPro)(i.e. pseudo-proline), or hydrogen. If P4 is a hydroxyl-protectinggroup, the process also comprises the step of cleaving thehydroxyl-protecting group P₄ from the Degarelix precursor. Theprotecting group P₄ is preferably selected in such a way that thiscleavage step can be carried out simultaneously with the cleavage of theamino-protecting group Pε. This is for example the case if both P₄ andPε are BOC.

In a second aspect, the invention also relates to a liquid-phase processfor preparing a protected Degarelix precursor having the formula(P₄)(Pε)Ac-AA₁-AA₁₀-NH₂ or a pharmaceutically acceptable salt or solvatethereof, comprising the step of coupling (P₄)Ac-AA₁-AA₄ with(Pε)AA₅-AA₁₀NH₂ or coupling Ac-AA₁-AA₃ with (P₄)(Pε)AA₄-AA₁₀NH₂ in anorganic solvent comprising the two peptides, a peptide coupling reagentand an organic amine base dissolved therein wherein Pε is an aminoprotecting group. The peptides are represented below:

A third aspect concerns the liquid-phase process for preparing aDegarelix intermediate having the formula (P₄)Ac-AA₁-AA₄:

or a pharmaceutically acceptable salt or solvate thereof, comprising thestep of hydrolyzing a compound having the formula (P₄)Ac-AA₁-AA₄-R withan alkaline hydroxide, wherein R represents a carboxyl protecting group,preferably C₁-C₄ alkyl or benzyl:

A fourth aspect concerns a process for preparing the compound(P₄)Ac-AA₃-AA₄-R by coupling Ac-AA₁-AA₃ with (P₄)AA₄-R or couplingAc-AA₁-AA₂ with (P₄)AA₃-AA₄-R, the peptides being represented below

In each of the formulae described above, AA₁ to AA₁₀, P₄ and P_(ε) havethe same meanings as in formula II, and R represents a carboxylprotecting group, preferably C₁-C₄ alkyl or benzyl

In a fifth aspect, the tetrapeptide (P4)Ac-AA₁-AA₄ is prepared not byliquid phase synthesis, but by solid phase synthesis. This inventionthus also relates to a solid-phase process for preparing a Degarelixintermediate having the formula (P4)Ac-AA₁-AA₄:

or a pharmaceutically acceptable salt or solvate thereof, comprising thesteps:

-   -   a) reacting (PN)AA2 with

to provide

-   -   b) removal of PN from

to provide

-   -   c) reacting (PN)AA1 with

to provide

-   -   d) if PN is not acetyl, removal of PN from

to provide

and subsequently acetylating

to provide

and

-   -   e) cleaving

to provide (P4)AC-AA₁-AA₄.wherein P4 is H or a hydroxyl protecting group on AA4, and PN is anamino protecting group.

A sixth aspect of the invention concerns liquid-phase process forpreparing the hexapeptide (Pε)AA₅-AA₁₀NH₂ comprising the coupling of(Pε)AA₆-AA₁₀NH₂ and (P_(X))AA₅, wherein P_(X) is an amino protectinggroup and AA₅ to AA₁₀ and Pε have the same meaning as above, to provide(P_(X))(Pε)AA₅-AA₁₀NH₂, and cleaving Px with TFA to provide(Pε)AA₅-AA₁₀NH₂, the peptides being represented below:

A seventh aspect of the invention concerns a liquid-phase process forpreparing the hexapeptide (Px)(Pε)AA₅-AA₁₀NH₂ comprising the coupling of(Px)AA₅-AA₇ with (Pε)AA₈-AA₁₀NH₂.

An eighth aspect of the invention concerns processes for purifyingDegarelix, such as by preparative HPLC using PLRP—S and/or C8 and C18columns.

It should be understood that in the process of preparing Degarelixaccording to this invention, any of the above process steps may becombined. For example, this invention also embodies a process in which(P₄)Ac-AA₁-AA4 is first prepared from (P₄)Ac-AA₁-AA₄-R according to thethird aspect of the invention or according to the fifth aspect of theinvention before being coupled with (Pε)AA₅-AA₁₀-NH₂ to form theprotected precursor (P₄)(Pε)Ac-AA₁-AA₁₀-NH₂ according to the secondaspect of the invention. The precursor (P₄)(Pε)Ac-AA₁-AA₁₀-NH₂ formed bysuch a process may then be deprotected according to the first aspect ofthe invention ultimately giving a single process for preparing Degarelixthat incorporates the first, second and third or fifth aspects of theinvention.

Naturally, any of the purification steps for Degarelix that aredescribed herein may be incorporated into any process in which Degarelixis the final product.

FIGURES

FIG. 1. Liquid phase preparation of the tetrapeptide Ac-AA₁-AA₄

FIG. 2. Preparation of the Pε protected hexapeptide AA₅-AA₁₀NH₂ where Pεis Fmoc.

FIG. 3. Segment condensation and deprotection to yield Degarelix.

FIG. 4: Solid phase preparation of the tetrapeptide Ac-AA₁-AA₄ with apseudoproline protecting group on AA4.

FIG. 5: Solid phase preparation of the tetrapeptide Ac-AA₁-AA₄ with atBu protecting group on AA4.

FIG. 6: Solid phase preparation of BOC-protected AA₅-AA₇

FIG. 7: Solid phase preparation of Fmoc-protected AA₈-AA₁₀NH₂.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in more detail.

Deprotection of the Degarelix Precursor

In a first aspect, the present invention relates to a liquid-phaseprocess for preparing Degarelix having the formula Ac-AA₁-AA₁₀-NH₂ or apharmaceutically acceptable salt or solvate thereof. The processcomprises the step of cleaving an ε-amino protecting group Pε from aDegarelix precursor according to formula (P₄)(Pε)AA₁-AA₁₀ in an organicsolution comprising the precursor and a cleaving agent dissolvedtherein.

In this case, Pε is any side chain protecting group known in the artsuch as those described in E. Gross & J. Meienhofer, The Peptides:Analysis, Structure, Biology, Vol. 3: Protection of Functional Groups inPeptide Synthesis (Academic Press, N.Y., 1981), Suitable examplesinclude 9-fluorenylmethyloxycarbonyl (Fmoc), CBZ, and substituted CBZ,such as, e.g., p-chlorobenzyloxycarbonyl, p-6-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, and p-methoxybenzyloxycarbonyl,o-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,2,6-dichlorobenzyloxycarbonyl, and the like; aliphatic urethane-typeprotecting groups, such as t-butyloxycarbonyl (Boc), t-amyloxycarbonyl,isopropyloxycarbonyl, 2-(p-biphenylyl)-isopropyloxycarbonyl, and thelike; cycloalkyl urethane-type protecting groups, such ascyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl; allyloxycarbonyl (Mac). Preferred protecting groupsare Fmoc, Boc and Alloc with Fmoc being most preferred.

If required, the hydroxyl group of Ser may also be protected, althoughthis is not preferred. In this case, P₄ is not hydrogen, but a hydroxylprotecting group such as for example, a C₄-C₆ alkyl (e.g. t-butyl,cyclohexyl), trityl, benzyl, a benzyl ether such as p-methoxybenzyl, orother substituted benzyls such as p-nitrobenzyl, p-chlorobenzyl,o-chlorobenzyl, 2,6-dichlorobenzyl, or (ψPro), (pseudoproline). If Seris protected, particularly preferred is t-butyl, benzyl and9-fluorenylmethyl ethers, t-butyl being most preferred. P4 is H, tBu, or(ψPro), preferably tBu or (ψPro),

The cleaving agent used to remove the ε-amino protecting group or theSer hydroxyl protecting group depends on the nature of the protectinggroup and are well known in the art. In a preferred embodiment, the samecleaving agent is used for both the ε-amino protecting group and the Serhydroxyl protecting group, if present.

Preferred cleaving agents for the Ser hydroxyl protecting group are:

-   -   trifluoracetic acid (TFA), HCl, or methanesulfonic acid,        particularly for t-butyl ether as a protecting group    -   H₂/Pd—C, HF, or trifluoromethane sulfonic acid, particularly for        benzyl ether as a protecting group, and    -   SiCl₄/anisol, particularly for 2-(methylsulfinyl)benzylether as        a protecting group;

Preferred cleaving agents for the ε-amino protecting group are:

-   -   trifluoracetic acid (TFA), HCl, or methanesulfonic acid,        particularly for t-butyl carbamates as protecting group    -   H₂/Pd—C, HF, or trifluoromethane sulfonic acid, particularly for        benzyl carbamates as protecting group, and    -   Piperidine, DBU and DEA, particularly for Fmoc as protecting        group

Preferred solvents include DCM, DMF, NMP, dioxane, EtOH, Neat HF, andTFA,

Particularly preferred are the different cleavage conditions indicatedin the following table 1:

TABLE 1 Cleavage conditions Protecting group Protected Abbreviation Namegroup Cleavage reagent Solvent t-Bu t-Butyl ethers and esters —OH andTFA DCM —CO₂H HCl Dioxane Methanesulfonic DCM acid Bzl Benzyl ethers andesters —OH H₂/Pd—C EtOH/water and HF Neat —CO₂H Trifluoromethane- DCMsulfonic acid MsOb 4-(Methylsulfinyl)-benzyl —OH SiCl₄/anisol TFA etherTce 2,2,2-Trichloroethyl esters —CO₂H Zn AcOH/H₂O Cbz or ZBenzyloxycarbonyl —NH₂ H₂/Pd—C EtOH/Water/acid HF Neat Trifluoromethane-DCM sulfonic acid Boc tert-Butoxy-carbonyl —NH₂ TFA DCM HCl DioxaneMethanesulfonic DCM acid Fmoc 9-Fluorenylmethoxy- —NH₂ piperidine DMFcarbonyl DBU (1,8- DMF diazabicyclo[5.4.0]- undec-7-ene) DEA(diethylamine) DMF Trt Trityl (Trt) —OH 1% TFA—DCM DCM —NH₂ TBDMSTert-butyl-dimethyl-silyl —OH TFA THF ACOH—THF—H₂O (3:1:1), 18 h 0.1MTBAF in THF Cyclohexyl Cyclohexyl —OH HF or TFSMA Neat HF or (CHX orCH_(x)) DCM Troc 2,2,2-Trichloroethoxy- —NH₂ Zn AcOH/H₂O carbonylReference: Chem. Rev. 2009, 109, 2465-2504 (by Albert Isidro-Llobet etal.)

Typically, a cleaving agent such as piperidine is dissolved in anorganic solvent such as DMF, NMP under an inert atmosphere such as N₂ orargon and cooled to a temperature between −20 and 0° C., preferably −10and −2° C., e.g. about −5° C. The protected intermediate (Pε)AA₁-AA₁₀ isadded and the reaction mixture is then stirred at a temperature ofbetween −20 and 25° C., preferably −10 and 10° C. and more preferably 0and 5° C. When the protecting group has been removed (preferably theyield is >95% yield, most preferably >99%), the crude Degarelix can beprecipitated, filtered and then washed with ether. For example, thecrude Degarelix can be precipitated by adding it to ether, such asmethyl t-butyl ether (MTBE) or DIPE, and stirring for 10 to 30 minutes.The precipitate can then be washed with ether (preferably DIPE).Subsequently, the solid may be taken up in e.g. ethyl acetate andstirred for some time at room temperature. The fine solid obtained maythen be filtered, washed (e.g. with ethyl acetate) and dried undervacuum.

It has been found that extended reaction periods are not detrimental tothe quality of the reaction and that no significant (<0.03% yield asdetermined by HPLC) increase in hydantoin impurity is observed if thereaction is allowed to proceed for up to 24 hours. Furthermore,particularly in the case of piperidine as a cleaving agent, no hydantoinimpurity is observed if the reaction is performed in the presence of 5Vol % water per volume of solvent (e.g. 0.1 ml of water per 2 ml solventsuch as DMF) for up to 20 hours. This demonstrates the robustness ofthis deprotecting reaction for the PPS of Degarelix.

4+6 coupling and 3+7 coupling

In a second aspect, the invention relates to a liquid-phase process forpreparing the protected Degarelix precursor having the formula(P₄)(Pε)AA₁-AA₁₀ or a pharmaceutically acceptable salt or solvatethereof. This process may comprise the step of coupling a tetrapeptideintermediate according to formula (P₄)Ac-AA₁-AA₄ with a hexapeptideintermediate according to formula (Pε)AA₅-AA₁₀. It may also comprise thestep of coupling a tripeptide intermediate according to formulaAc-AA₁-AA₃ with a heptapeptide intermediate according to formula(P₄)(Pε)AA₄-AA₁₀. In either case, the protecting group Pε may be anyε-amino protecting group as discussed previously. The hydroxyl group ofSer may also be protected if required (i.e., in this case P4 is nothydrogen, but a hydroxyl protecting group). The coupling reaction isperformed in an organic solution where the two peptides, a peptidecoupling reagent and an organic amine base are dissolved therein. Apeptide coupling additive may also be present.

The organic solvent, peptide coupling reagent, peptide coupling additiveand organic amine base may be any of those known in the art of LPPS.

Typical organic solvents are THF, NMP (N-methylpyrrolidone), Davi, DMF,DMSO, and mixtures thereof.

Typical peptide coupling reagents are one or more ofo-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),o-(benzotriazol-1-0)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), o-(benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU),benzotriazole-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP),benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate(PyBOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP—Cl),bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP),iso-butylchloroformate (IBCF), 1,3 dicyclohexylcarbodiimide (DCC),1,3-diisopropyl-carbodiimide (DIC),1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSCDl),N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ),isopropylchloroformate (IPCF),2-(5-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TNTU), propane phosphonic acid anhydride (PPAA) and2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU).Preferred coupling reagents are DIC, HATU, HBTU, and BOP.

Typical peptide coupling additives are3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt),1-hydroxy-1H-benzotriazole (HOBt), 6-chloro-HOBt, and1-hydroxy-7-azabenzotriazole (HOAt). Particularly preferred are HOBt andHOAt.

Typical organic amine bases are NMM, DIPEA, TEA, and collidine.Particularly preferred is DIPEA.

Particularly preferred is the combined use of HATU, HOAt, and DIPEA.Another preferred embodiment relates to the combined use of DIC,6-chloro-HOBt, optionally in combination with copper salts.

Surprisingly, it has been found that the choice of organic solvent,peptide coupling reagent/additive and organic amine base has an effecton the yields of the desired products and on the racemisation of the Seramino acid in the polypeptide backbone.

For instance, while THF, NMP, DCM, DMF and mixtures thereof can be usedas solvents for these coupling reactions, the use of DMF, either aloneor in a mixture (e.g. with DCM), increases the yield of the desiredfinal product while at the same time reducing any D-Ser impurity. Theuse of DMF apparently increases the yield and reduces D-Ser impurities.The activation is rapid in a polar solvent such as DMF but slow in anon-polar solvent such as DCM. The HATU/HOAt combination leads to ahighly efficient coupling partly because of rapid activation by a basesuch as DIEA or NMM followed by rapid coupling in the presence of thesame base in a polar solvent like DMF. The effect of different solventsis illustrated in Table 2.

TABLE 2 Screening of different solvents during the coupling of AA1-AA4and AA5-AA10 segment Coupling Purity by HPLC (%) Reagent & Yield ProductImpurity Entry Solvent additive (%) CDEG-1 (D-ser) Remarks 1 THF EDC•HCl35 62.42 16.29 NMM as a base; CLEU-15 content 26.5% 2 NMP EDC•HCl 3562.42 16.29 NMM as a base; CLEU-15 content 0.17% 3 DCM-DMF EDC•HCl 5347.81 10.39 CLEU-15 content 25.54% 4 DMF EDC•HCl 65 72 9.2 Collidine asa base 5 DMF HATU/HOAt 74 84.4 1.91 3.0 eq. of DIEA is used 6 DMFHATU/HOAt 74 85.3 1.44 3.0 eq. of DIEA is used 7 DMF HATU/HOAt 87 83.651.44 3.0 eq. of DIEA is used

The choice of coupling reagent and additive also has a large effect onthe yield and degree of racemisation. Coupling reactions using HBTU,HCTU, TBTU, BOP and HATU were performed and found with HBTU and HATUgiving the best overall yields. However, it was found that couplingreactions using BOP or HATU as coupling reagent led to much lessracemisation of the Ser amino acid.

The addition of a coupling additive significantly improves the yield ofthe desired polypeptide. In many cases the coupling additive alsoreduces the degree of racemisation of the Ser amino acid even furtherthus leading to a product with fewer impurities. Combinations ofcoupling reagent and additive which increased yield are TBTU/HOAt,HATU/HOAt and HATU/HOBt. Surprisingly, the combination of TBTU/HOAt orHATU/HOAt increased yield while at the same time reducing racemisation,with HATU/HOAt performing best out of all the combinations tested.

The effect of different coupling agents and additives is illustrated inthe following Tables 3 and 4.

TABLE 3 Screening of different coupling reagents to control theracemization Coupling Purity by HPLC (%) Reagent & CDEG-1 Impurity EntryBase additive Yield (%) CDEG-1 (D-ser) Remarks 1 NMM EDC•HCl/HOBt 67 6520 Racemization is more 2 NMM EDC•HCl/HOBt 67 65 18.9 Racemization ismore 3 DIEA TBTU 39 67.93 13.34 1.3% of CLEU-15 remains 4 DIEA HBTU 5354.9 20.49 1.9% of CLEU-15 remains 5 DIEA EDC•HCl 51 — — 68.9% ofCLEU-15 remains 6 NMM HBTU 78 61.42 15.16 9.8% of CLEU-15 remains 7 NMMHCTU 60 51.63 15.92 7.32% of CLEU-15 remains 8 NMM TBTU 53 62.78 14.733.75% of CLEU-15 remains 9 NMM BOP 51 46.3 8.92 11.93% of CLEU-15remains 10 NMM HATU 74 65.97 10.62 4.52% of CLEU-15 remains 11 DIEATBTU/HOAt 98 76.26 6.8 0.14% of CLEU-15 remains 12 DIEA DIC/HOAt — 20.491.18 45.9% of CLEU-15 remains 13 DIEA HATU/HOAt 74 83.16 2.9 Nounreacted CLEU-15 14 NMM HATU/HOAt 81 64.0 13.32 7.1% of CLEU-15 remains15 NMM HATU/HOBt 89 65.0 13.14 4.35% of CLEU-15 remains 16 NMMEDC•HCl/HOAt 74 70.38 13.66 No unreacted CLEU-15 17 DIEA HATU/HOAt 7979.79 2.3 This reaction was repeated in larger scale 18 DIEA TBTU/HOAt84 75.8 7.2 Reaction was repeated in larger scale. 19 DIEA HATU/HOAt 7484.4 1.91 Reaction was repeated in larger scale. 20 DIEA HATU/HOAt 7485.3 1.44 Reaction was repeated in larger scale. 21 DIEA HATU/HOAt 87 841.44 Reaction was repeated in larger scale. 22 DIEA HATU/HOAt 79 84.51.37 Reaction was repeated in larger scale.

TABLE 4 Screening of different additives to control the racemizationduring the coupling of AA1-AA4 and AA5-AA10 segment Coupling CDEG-1Purity by HPLC (%) Entry Reagent Additive Base Yield (%) CDEG-1 (D-ser)Remarks 1 TBTU HOAt DIEA 98 76.26 6.8 0.14% of CLEU-15 remains 2 DICHOAt DIEA — 20.49 1.18 45.9% of CLEU-15 remains 3 HATU HOAt DIEA 7483.16 2.9 Completion of reaction 4 HATU HOAt NMM 80 64.0 13.32 7.1% ofCLEU-15 remains 5 HATU HOBt NMM 89 65.0 13.14 4.3% of CLEU-15 remains 6EDC•HCl HOAt NMM 74 70.38 13.66 No unreacted CLEU-15 7 HATU HOAt DIEA 7979.79 2.3 Repeat reaction 8 TBTU HOAt DIEA 84 75.8 7.2 Repeat reaction 9HATU HOAt DIEA 74 84.4 1.91 3.0 eq. of base is used 10 HATU HOAt DIEA 7485.3 1.44 3.0 eq. of base is used 11 HATU HOAt DIEA 87 83.65 1.44 3.0eq. of base is used 12 EDC•HCl HOBt NMM 67 65 20 Racemization is more 13EDC•HCl HOBt NMM 64 65 17.9 Racemization is more 14 EDC•HCl HOBt NMM 6765 18.9 Racemization is more 15 HOSu NMM — — — Reaction did not proceed

The choice of organic amine base also affects the reaction. For thepresent invention, NMM and DIEA are preferred as they allow the desiredpolypeptide to be obtained in the best yields. DIEA is more preferredsince this base reduces the degree of Ser racemisation. It has also beenfound that the amount of base affects the reaction. When a base such asDIEA is used, it was found that the more base present, the lower theyield and higher the degree of racemisation. For example, sixequivalents of base (with respect to A51-A10) lead to a two-foldincrease of the racemisation product as when three equivalents of baseare used. Thus, it is preferred to use 1-5 equivalents of base, morepreferably 2-4 equivalents of base and most preferably 2.5 to 3.5equivalents of base in these coupling reactions. The effect of differentbases and their amounts is shown in the following tables 5 and 6.

TABLE 5 Screening of different bases to control the racemization duringthe coupling of AA1-AA4 and AA5-AA10 segment Coupling Yield (%) Purityby HPLC (%) Entry Reagent Additive Base CDEG-1 CDEG-1 (D-ser) Remarks 1TBTU HOAt DIEA 98 76.26 6.8 0.14% of CLEU-15 remains 2 DIC HOAt DIEA —20.49 1.18 45.9% of CLEU-15 remains 3 HATU HOAt DIEA 74 83.16 2.9 NoCLEU-15 remains 4 HATU HOAt NMM 80 64.0 13.32 7.1% of CLEU-15 remains 5HATU HOBt NMM 89 65.0 13.14 4.35% of CLEU-15 remains 6 EDC•HCl HOAt NMM74 70.38 13.66 No CLEU-15 remains 7 HATU HOAt DIEA 79 79.79 2.3 8 TBTUHOAt DIEA 84 75.8 7.2 9 HATU HOAt DIEA 74 84.4 1.91 3.0 eq. of base isused 10 HATU HOAt DIEA 74 85.3 1.44 3.0 eq. of base is used 11 HATU HOAtDIEA 87 83.65 1.44 3.0 eq. of base is used 12 EDC•HCl HOBt Collidine 6572 9.2 13 EDC•HCl HOBt NMM 67 65 20 Racemization is more 14 EDC•HCl HOBtNMM 64 65 17.9 Racemization is more 15 EDC•HCl HOBt NMM 67 65 18.9Racemization is more

TABLE 6 Screening of base equivalence to control the racemization duringthe coupling of AA1-AA4 and AA5-AA10 segment DIEA Qty Coupling Reagent &CDBG-1 Purity by HPLC (%) Entry (eq.) additive Yield (%) Product CDEG-1Impurity (D-ser) 1 6.0 HATU/HOAt 62 74.19 1.5 2 5.0 HATU/HOAt 69 79.003.88 3 4.5 HATU/HOAt 68 84.12 1.18 4 4.0 HATU/HOAt 70 82.64 1.19 5 3.0HATU/HOAt 74 84.4 1.91 6 3.0 HATU/HOAt 74 85.3 1.44 7 3.0 HATU/HOAt 8783.65 1.44

The temperature that the coupling reaction is performed also influencesthe yield and the degree of racemisation of the final product. It wasfound that a reaction carried out at −15° C. gives higher yield, higherpurity and less racemisation of the final product than the equivalentreaction carried out at −5° C. Thus, it is preferable to carry out thesecoupling reactions at temperatures lower than −5° C., preferably lowerthan −10° C. and most preferably at −15° C. or lower. The reaction timeof these coupling reactions is usually 2-3 hours.

It should be noted that by controlling the reaction temperature and theamount of base added, it is also possible to reduce any hydantoinimpurity formation. The hydantoin content is preferably less than 0.5wt. %, more preferably less than 0.3 wt. %. Thus, it is preferred to use2.5 to 3.5 equivalents of base at temperatures of −10° C. or lower inthese reactions.

Finally, the order of addition of the various reagents also plays a rolein the final yield, purity and amount of racemisation. If the peptidesand coupling additive are first dissolved in the organic solvent beforethe coupling reagent and the organic amine are added, the overall yieldof the desired product is significantly higher. Furthermore, the amountof racemisation is drastically reduced.

Fragment (P₄)Ac-AA₁-AA₄

The present invention provides different methods for preparing(P₄)Ac-AA₁-AA₄.

In a third aspect, the invention relates to a liquid-phase process forpreparing a Degarelix intermediate having the formula (P₄)Ac-AA₁-AA₄:

or a pharmaceutically acceptable salt or solvate thereof, wherein P₄ ishydrogen or a hydroxyl-protecting group, preferably hydrogen.

When preparing (P₄)Ac-AA₁-AA₄, an ester having the formula(P₄)Ac-AA₁-AA₄-R is first prepared, wherein R is a carboxyl protectinggroup, such as a benzyl group, preferably however a C₁-C₄ alkyl group.Normally, a benzyl ester of serine is used, the benzyl group being thenremoved by hydrolysis with tetrabutylammonium hydroxide (see for exampleWO 99/26964). However, it was found that in the preparation ofDegarelix, the tetrabutylammonium ions were not removed completelyduring subsequent operations and were carried through to the finalproduct. This problem was overcome by using a C₁-C₄ alkyl ester ofserine (e.g. serine methyl ester). It was found that the alkyl estercould be easily hydrolyzed using an alkali hydroxide such as LiOH. Theyield and quality of the tetrapeptide was not affected by this changeand the problem of tetrabutylammonium ion impurities was eliminated.

For example, a compound according to formula (P₄)Ac-AA₁-AA₄-R may besuspended in an organic solvent such as THF and then stirred and cooledto a temperature of between −20 and 5° C., and more preferably −5 and 0°C. An aqueous solution of LiOH is then added to the cooled solution. Theaqueous LiOH is added at a rate that maintains the temperature of thecooled solution in the range of −5 to 0° C. or below. The solution(oftentimes turbid) is stirred for up to 12 hours, preferably up to 3hours, before being added, with good stirring, to water with atemperature of 5° C. or below, or preferably a mixture of ice and water.Any precipitate at this point is removed by filtration. The pH is thenadjusted to pH 4.1-4.3, preferably about 4.2 using any known pHadjusting agent. Preferred is HCl, for example 2M HCl. The precipitatethat forms after adjusting the pH is collected by filtration. Theprecipitate can be further purified by washing it with water, and/orstirring a slurry of it in refluxing MeOH and/or a MeOH/MeCN mixturebefore collecting it by filtration and then drying it to yield(P₄)Ac-AA₁-AA₄.

A fourth aspect of the invention concerns a process for preparing thecompound (P₄)Ac-AA₁-AA₄-R by coupling Ac-AA₁-AA₃ with (P₄)AA₄-R orcoupling Ac-AA₁-AA₂ with (P₄)AA₃-AA₄-R, wherein R is a C₁-C₄-alkyl andP₄ is hydrogen or a hydroxyl-protecting group, preferably hydrogen. Forthe coupling reaction, essentially the same reagents and conditions asthose described above can be used.

In a fifth aspect, this invention relates to a solid-phase process forpreparing a Degarelix intermediate having the formula (P4)Ac-AA₁-AA₄:

or a pharmaceutically acceptable salt or solvate thereof, comprising thesteps:

-   -   a) reacting (PN)AA2 with

to provide

-   -   b) removal of PN from

to provide

-   -   c) reacting (PN)AA1 with

to provide

-   -   d) if PN is not acetyl, removal of PN from

to provide

and subsequently acetylating

to provide

and

-   -   e) cleaving

to provide (P4)AC-AA₁-AA₄.wherein P4 is H or a hydroxyl protecting group on AA4, and PN is anamino protecting group.

PN is preferably Fmoc, which is preferably removed with piperidine/NMP.

P4 is preferably tBu or (WPro). Particularly preferred is thecombination of (ψPro) for P4 and Fmoc for PN.

Each coupling step is preferably carried out in a manner known per se,preferably however using HATU (or HBTU) and DIPEA and couplingadditives.

The starting material

starting can be prepared by coupling (PN, P4)AA₃-AA₄ to a resin, forexample to a 2-CITrt resin, and then removing PN, or by coupling (PN,P4)AA₄ to

to obtain

removing PN, and then reacting (PN)AA₃ with

to provide

and then removing PN. This is illustrated in FIGS. 4 and 5.

Suitable resins

include trityl, 2-CITrt and SASRIN.

In the case P4 is (ψPro) and PN is Fmoc, (PN, P4)AA₃-AA₄ can be preparedfollowing J. Am, Chem. Soc. 1996, 118, 9218-9227. That is, Fmocprotected AA3 is activated; reacted with serine or a salt thereof, andsubsequently reacted with acetone or acetone dimethylketal, asillustrated below. Fmoc-D3 PaI-Ser((ψ^(Me,Me)Pro)-OH is particularlypreferred as (PN, P4)AA₃-AA₄.

Fragment (P_(X))(Pε)AA₅-AA₁₀NH₂

A sixth aspect of the invention concerns liquid-phase process forpreparing the hexapeptide (Pε)AA₅-AA₁₀NH₂ comprising the coupling of(Pε)AA₆-AA₁₀NH₂ and (P_(X))AA₅, wherein P_(X) is an amino protectinggroup and AA5 to AA10 and Pε have the same meaning as above, to provide(P_(X))(Pε)AA₅-AA₁₀NH₂, and cleaving Px with TFA to provide(Pε)AA₅-AA₁₀NH₂.

A seventh aspect of the invention concerns a liquid-phase process forpreparing the hexapeptide (Pε)AA₅-AA₁₀NH₂ by coupling (P5)AA₅-AA₇ with(Pε)AA₈-AA₁₀NH₂ to provide (P5, Pε)AA₅-AA₁₀NH₂, and subsequentlycleaving P5 to provide (Pε)AA₅-AA₁₀NH₂ (wherein P5 is anamino-protecting group on AA5).

P5 protecting group is preferably BOC. Pε is preferably9-fluorenylmethyloxycarbonyl (Fmoc). The coupling reaction is preferablycarried out in the presence of HATU.

(P5)AA₅-AA₇ and (Pε)AA₅-AA₁₀NH₂ are preferably synthesized by solidphase peptide synthesis, e.g. as illustrated in FIGS. 6 and 7,respectively. The following combinations of P5 and Pε are preferred:

P5 Pε Boc Fmoc Cbz Boc Troc Boc

That is, (P5)AA₅-AA₇OH can be prepared by coupling protected AA7 to aresin; removing the protecting group (e.g. Fmoc); reacting protected AA6with the obtained product; removing the protecting group (e.g. Fmoc);reacting protected AA5 (e.g. BOC protected) with the obtained product;and cleavage from the resin. This is illustrated in FIG. 6.

(Pε)AA₅-AA₁₀NH₂ can be prepared by coupling protected AA10 to a resin,e.g. a Rink amide resin; removing the protecting group (e.g. Fmoc);reacting protected AA9 with the obtained product; removing theprotecting group (e.g. Fmoc); reacting protected AA8 (preferablyBoc-protected on the alpha-amino group and Fmoc-protected on the sidechain) with the obtained product; removing the alpha-amino protectinggroup; and cleavage from the resin. This is illustrated in FIG. 7.

(Pε)AA₈-AA₁₀NH₂ can also be prepared by reacting AA10 NH₂ with protected(e.g. BOC) AA9; removing the protecting group; reacting AA9-AA10 NH₂with protected AA8 (preferably Boc-protected on the alpha-amino groupand Fmoc-protected on the side chain); and removing the protecting groupon the alpha-amino group.

In an eighth aspect, the invention relates to the purification ofDegarelix. The purification can be carried out in a manner known to theskilled person, e.g. by preparative chromatography.

For example, first purification of Degarelix is achieved with a PLRP—Sstationary phase, pH 2.25 using TEAP as a buffer and MeCN (75:25) asmobile phase. Purity of up to 95% can be obtained with this step. Ifrequired, a second purification can be carried out using a combinationof C8 and C18 columns (e.g. Zorbax) to achieve purity of 99% and above.

EXPERIMENTAL

The following examples are intended to illustrate a process for the LPPSsynthesis of Degarelix. Reference is made to FIGS. 1 and 2 for thestructures of each peptide and polypeptide described herein.

CLEU-2:

L-Cbz-proline (50.0 g) was dissolved in 2-propanol (500 ml{circumflexover (=)}10 V) and the solution was cooled to 15° C. N-Methylmorpholine(25 ml{circumflex over (=)}0.5 V) was then added slowly. After stirringthe solution for 15 minutes, 28.49 g (1.04 eq.) iso-butyl chloroformatewas added dropwise at −15° C. A solution of D-ala-NH₂.HCl (27.48 g, 1.1eq) and NMM (25 ml{circumflex over (=)}0.5 V) in water (250ml{circumflex over (=)}5 V) was added to the reaction mixture at −15° C.The mixture was stirred for 30 minutes at the same temperature and thenwarmed to 25° C. and stirred for 3-5 hrs. The reaction mixture wasquenched by adding ethyl acetate (1000 ml{circumflex over (=)}20 V) andwater (10 V) containing NaCl (25 g) and NaHCO₃ (25 g). The organiclayers were separated, washed with water (2×500 ml{circumflex over(=)}10 V) and then dried over sodium sulphate. The organic layer wasconcentrated to 4 volumes under vacuum below 40° C. The solution wasdiluted with ethyl acetate (250 ml{circumflex over (=)}5 V) and n-hexane(375 ml{circumflex over (=)}7.5 V) was added dropwise to obtain a whitesolid. The solid was filtered off and dried to afford the product.

Output: 35.2 g; 54.7%; [α]₂₅ ^(D): −13.0° [CHCl₃, Literature report:−11.2° (U.S. Pat. No. 5,710,246)]). HPLC purity: 99.44%

CLEU-4:

CLEU-2 (19.5 g) was taken in 2-propanol (129 ml{circumflex over (=)}6.5V) into a Parr hydrogenation flask and a solution of p-toluenesuiphanicacid in water (20 ml{circumflex over (=)}1.0 V) was added to it. 10%Pd/C (5% w/w) was added to the reaction mixture and the mixture thenhydrogenated at 40 psi for 2 hrs. When TLC showed the disappearance ofthe starting material, the catalyst was filtered and washed with2-propanol (79 ml{circumflex over (=)}4 V) and water (8.75 ml{circumflexover (=)}0.5 V). The filtrate was concentrated under vacuum and strippedoff with acetonitrile (4×254 ml{circumflex over (=)}13 V). The residuewas taken in a flask and acetonitrile (215 mL{circumflex over (=)}11 V)was added followed by Boc-Lys(Cbz)-OH (25.5 g{circumflex over (=)}1.1equiv) and HOBt (9.9 g{circumflex over (=)}1.2 equiv). The suspensionwas cooled to −5° C. and NMM (14.95 g{circumflex over (=)}2.45 equiv)was added slowly. Finally, a solution of EDC.HCl (15.3 g{circumflex over(=)}1.3 equiv) in acetonitrile (120 ml{circumflex over (=)}6 V) wasadded. The reaction mixture was then warmed to 25° C. and stirred for10-12 hrs at the same temperature. The solvent was removed under vacuumbelow 40° C. and diluted with water (120 ml{circumflex over (=)}6 V).The product was extracted with ethyl acetate (878 ml{circumflex over(=)}45 V), water (120 ml{circumflex over (=)}6 V), and 10% sodiumcarbonate (110 ml{circumflex over (=)}5.7 V). The aqueous layer wasextracted with ethyl acetate (2×430 ml{circumflex over (=)}2×22 V) andthe organic layers combined and washed with 10% citric acid solution(2×105 ml), 10% sodium carbonate solution (2×110 ml{circumflex over(=)}2×5 V), water (120 ml{circumflex over (=)}6 V) and dried over sodiumsulphate. The organic layer was concentrated to 10-12 volumes undervacuum below 40° C., stripped off with ethyl acetate (3×20 V) andmaintained the final 10-12 volumes in each stripping. N-hexane (14 V)was added dropwise to the concentrate mass to get a white solid. Thesolid was filtered off and dried to afford the product.

Output 29.2 g; Yield: 86.6%; Purity 99.18%, [α]₂₅ ^(D): −26.0° (c 1,CHCl₃)

CLEU-5:

CLEU-4 (16.3 g) was taken in a mixture of methanol (163 ml{circumflexover (=)}10 V), acetone (22 ml{circumflex over (=)}1.4 V) in a stainlesssteel Parr hydrogenation flask. 10% Pd/C (10% w/w) was added and themixture was hydrogenated (60 psi) for 8-12 hrs at 25° C. After thestarting material had disappeared (TLC), the catalyst was filteredthrough celite and was washed with methanol (163 ml{circumflex over(=)}10 V). The filtrate was concentrated under vacuum below 40° C. andthe residue was stripped off with ethyl acetate (3×143 ml{circumflexover (=)}3×9 V). The residue was then taken in ethyl acetate (51ml{circumflex over (=)}3 V) and n-hexane (20.4 ml{circumflex over(=)}1.25 V) added. The mixture was stirred for 2-3 hrs to obtain a freesolid. The solid was filtered, washed with n-hexane (38 ml{circumflexover (=)}2 V), and dried under vacuum below 40° C.

Output: 12.3 g; Yield: 90.8%; Purity: 98.9%

CLEU-6:

CLEU-5 (12.2 g) was added to THF (40 ml{circumflex over (=)}3.3 V) andthe mixture was cooled to 0° C. 10% sodium carbonate solution (34ml{circumflex over (=)}2.7 V) was added to the mixture over 20 minutes.Fmoc-Cl (8.63 g{circumflex over (=)}1.2 equiv) in THF (12.2ml{circumflex over (=)}1.0 V) was then added slowly over 15-20 minutesat 0° C. The reaction mixture was stirred for 1 hr at same temperatureand diluted with water (134.2 ml{circumflex over (=)}11 V). The productwas extracted with ethyl acetate (269 ml{circumflex over (=)}22 V). Theorganic layer was washed with water (134.2 ml{circumflex over (=)}11 V),10% citric acid solution (2×134 ml{circumflex over (=)}2×11 V) and water(134.2 ml{circumflex over (=)}11 V). The organic layer was concentratedunder vacuum below 40° C. and the crude product was purified by columnchromatography.

Output: 13.3 g; Yield: 73.2%; Purity: 98.2%

CLEU-7:

CLEU-6 (9.0 g) was charged to a TFA (61 ml{circumflex over (=)}6.75 V)and m-cresol (0.61 ml) solution at −5° C. The reaction mixture wasstirred for 2 hrs at 0° C. and then concentrated under vacuum below 35°C. Traces of TFA were removed by co-distillation with toluene (2×45 ml).The product was crystallized from a mixture of MTBE (9 ml{circumflexover (=)}1 V), and DIPE (90 ml{circumflex over (=)}10 V). The solid wasfiltered off under nitrogen, washed with DIPE (180 ml{circumflex over(=)}20 V) and dried under vacuum to get pure CLEU-7.

Output: 8.8 g; Yield: 90.4%; Purity: 98.3%.

Comment: CLEU-7 material is hygroscopic in nature and thus should behandled with care.

CLEU-8:

CLEU-7 (8.5 g) was taken in acetonitrile (85 ml{circumflex over (=)}10V). Boc-Leu-OH (3.12 g{circumflex over (=)}1.1, equiv), HOBt (2.31g{circumflex over (=)}1.39 equiv) and NMM (1.4 ml{circumflex over(=)}1.03 equiv) were added to the solution. The solution was cooled to−2° C. and treated with NMM (1.4 ml{circumflex over (=)}1.03 equiv) andEDC.HCl (2.58 g{circumflex over (=)}1.1 equiv). The reaction mixture wasstirred for 2-3 hrs at 0° C. and the solvent was removed by distillationunder vacuum. 10% citric acid (85 ml{circumflex over (=)}10 V) and ethylacetate (213 ml{circumflex over (=)}25 V) were added to the residue. Theorganic layer was separated and washed with 10% citric acid solution(2×85 ml{circumflex over (=)}2×10.0 V), DM water (85 ml{circumflex over(=)}10 V), and 5% sodium bicarbonate solution (3×85 ml{circumflex over(=)}3×10.0 V) and again with DM water (85 ml{circumflex over (=)}10 V).Finally, the organic layer was dried over sodium sulphate andconcentrated under vacuum below 35° C. to obtain the crude product. Thecrude product was crystallized from MTBE (68 ml{circumflex over (=)}8 V)and n-hexane (34 ml{circumflex over (=)}4 V). The solid was dried undervacuum at below 35° C.

Output: 7.8 g; Yield: 81.2%; Purity: 97.5%.

CLEU-9:

CLEU-8 (7.5 g) was charged to a TFA (54 ml{circumflex over (=)}7.2 V)and m-cresol (0.27 ml) solution at −5° C. The reaction mixture wasstirred for 2.0 hrs at 0° C. and then concentrated under vacuum below35° C. Traces of TFA were removed by co-distillation with toluene (2×38ml the product was crystallized from MTBE (75 ml{circumflex over (=)}10V) and n-hexane (413 ml{circumflex over (=)}15 V). The solid wasfiltered under nitrogen, washed with n-hexane (37.5 ml{circumflex over(=)}5 V), and dried under vacuum to yield pure CLEU-9.

Output: 7.2 g; Yield: 94.8%; Purity: 97.2%

CLEU-12:

CLEU-9 (10.5 g) was taken in acetonitrile (158 ml{circumflex over (=)}15V) under nitrogen atmosphere. CMAP-5A (4.2 g{circumflex over (=)}1.0equiv), HOBt (2.11 g{circumflex over (=)}1.2 equiv) were added into thesuspension and the mixture was cooled to 0° C. EDC.HCl (2.73g{circumflex over (=)}1.1 equiv) was added into the suspension followedby slow addition of NMM (1.38 g{circumflex over (=)}1.05 equiv). Thereaction mixture was stirred for 0.5 hr at 0° C., warmed to ambientconditions and then stirring was continued for 3 hrs. The solvent wasremoved under vacuum below 35° C. and the residue was taken in a mixtureof 10% citric acid (105 ml{circumflex over (=)}10 V) and ethyl acetate(263 ml{circumflex over (=)}25 V). The organic layer was separated andwashed with 10% citric acid solution (105 ml{circumflex over (=)}10 V),DM water (105 ml{circumflex over (=)}10 V), 5% sodium bicarbonatesolution (3×105 ml{circumflex over (=)}3×10 V), and DM water (105ml{circumflex over (=)}10 V). The organic layer was dried over sodiumsulphate and concentrated under vacuum below 35° C. The crude productwas precipitated with n-hexane (84 ml{circumflex over (=)}8 V), washedwith n-hexane (2×84 ml{circumflex over (=)}2×8 V), and dried undervacuum at 35° C.

Output: 10.5 g; Yield: 80.7%; Purity: 92.1%

CLEU-13:

CLEU-12 (10.5 g) was charged to a TFA (78.8 ml{circumflex over (=)}7.5V) and m-cresol (0.4 ml) solution at −5° C. The reaction mixture wasstirred for 2 hrs at 0° C. and then concentrated under vacuum below 35°C. Traces of TFA were removed by co-distillation with toluene (2×52 ml).The residue was taken up in ethyl acetate (105 ml{circumflex over (=)}10V) and n-hexane (158 ml{circumflex over (=)}15 V) is added toprecipitate the product. The solid was filtered off under nitrogen andwashed with n-hexane (53 ml{circumflex over (=)}5 V). The compound wasdried under vacuum at 35° C.

Output: 9.6 g; Yield: 90.5%; Purity: 91.9%.

CLEU-14:

CLEU-13 (9.5 g) was dissolved in DMF (95 ml{circumflex over (=)}10 V)under a nitrogen atmosphere. NMM (1.0 g{circumflex over (=)}1.05 equiv),CSER-2 (3.95 g{circumflex over (=)}1.0 equiv) and HOBt (1.4 g{circumflexover (=)}1.1 equiv) were added to the solution and the reaction mass wascooled to 0° C. EDC.HCl (2.0 g{circumflex over (=)}1.1 equiv) and NMM(1.0 g{circumflex over (=)}1.05 equiv) were added subsequently into themixture. The reaction mass was stirred for 5 hrs at 0° C. and thenpoured into ice cooled water (950 ml{circumflex over (=)}100 V) andstirred for 30 minutes. The precipitated solid was filtered off andwashed with water (20 V), 10% citric acid (10 V), again with water (190ml{circumflex over (=)}20 V), 5% NaHCO₃ solution (95 ml{circumflex over(=)}10 V), and water (95 ml{circumflex over (=)}10 V). The product wasdried under vacuum below 35° C.

Output: 10.3 g; Yield: 84.40%; Purity: 90.4%.

CLEU-15:

CLEU-14 (10.0 g) was charged to a TFA (75 ml{circumflex over (=)}7.5 g)and m-cresol (0.37 ml) solution at −5° C. The reaction mixture wasstirred for 2 hrs at 0° C. and then concentrated under vacuum below 35°C. Traces of TFA were removed by co-evaporation with toluene (2×50 ml).The residue was taken up in ethyl acetate (100 ml{circumflex over (=)}10V) and n-hexane (60 ml{circumflex over (=)}6 V) was added to precipitatethe product. The solid was filtered off under nitrogen, washed withn-hexane (2×30 ml{circumflex over (=)}6 V) and then dried under vacuumat 35° C.

Output: 9.6 g; Yield: 95.0%; Purity: 91.5%.

CSER-2:

To a stirred solution of L-hydroorotic acid (23.4 g, 148 mmol) andN-hydroxysuccinimide (18.14 g{circumflex over (=)}1.1 equiv) in dry DMF(585 ml{circumflex over (=)}25 V) was added DIC (20.5 g{circumflex over(=)}1.1 equiv) with external ice water cooling. The reaction mixture wasstirred at room temperature for 13-14 hrs. The precipitate was filteredoff and the filtrate was evaporated. The oily residue was washed withdiisopropyl ether (94 ml{circumflex over (=)}4 V) and dissolved in dryDMF (293 ml{circumflex over (=)}12.5 V). N-Boc-L-4-aminophenylalanine(41.5 g{circumflex over (=)}1.0 equiv) was added to the above solution.DIEA (22.97 g{circumflex over (=)}1.2 equiv) was added at 0° C. and thereaction mixture was stirred for 22 hrs and the solvent then evaporated.The residue was mixed with water (702 ml{circumflex over (=)}30 V) andthe pH of the resulting suspension was adjusted to 9.0 with saturatedsodium bicarbonate solution. The precipitate of diisopropylurea wasfiltered off and the filtrate was washed with ethyl acetate (70.2ml{circumflex over (=)}3 V). The aqueous layer was acidified to pH 2.5with 6 N HCl and the resulting precipitate collected by filtration. Theproduct was obtained as a yellow solid.

Output: 40.0 g; Yield 64%; mp-270° C., [α]₂₅ ^(D)=+61.5 (c 1.0, 1%NaHCO₃); Purity 95%.

CBBC-2B:

Fragment-A (20.0 g) (purchased from Chirotech, UK) was dissolved in DMF(300 ml{circumflex over (=)}15 V) at 30-35° C. and then cooled −10° C.to 5° C. wherein HOBt (5.06 g{circumflex over (=)}1.1 equiv) was addedto the mixture, stirred for 30 minutes before L-serine methyl ester(5.29 g{circumflex over (=)}1.0 equiv) was added to the suspension. Themixture was stirred for 30 minutes, treated with NMM (7.23 g{circumflexover (=)}2.1 equiv) and then stirred for 30 minutes. EDC.HCl (7.18g{circumflex over (=)}1.1 equiv) was then added to the suspension andthe reaction mixture was stirred for 5 hrs at −5° C. to 0° C. Thereaction mixture was poured into chilled DM water (151 or 1500ml{circumflex over (=)}75 V) and stirred for 30 minutes. The product wasprecipitated by stirring at 0-5° C. After 1 hr the resulting solid wascollected by filtration. The filter cake was washed with water (200ml{circumflex over (=)}10 V), 10% citric acid (200 ml{circumflex over(=)}10 V), again with water (200 ml{circumflex over (=)}10 V), 5% NaHCO₃solution (200 ml{circumflex over (=)}10 V), water (200 ml{circumflexover (=)}10 V) and then the solid was dried under vacuum for 4 hrs. Theproduct was slurried in methanol (300 ml{circumflex over (=)}15 V) andstirred for 1 hr. The suspension was filtered and the cake washed withmethanol (100 ml{circumflex over (=)}5 V) then dried under vacuum at30-35° C. to a constant weight.

Output: 22 g; Yield: 93.8%.

CBBC-3B:

CBBC 2B (20.0 g) was suspended in THF (500 ml{circumflex over (=)}25 V)and then stirred and cooled to −5° C. to 0° C. To the cooled solutionwas added an aqueous solution of lithium hydroxide (3.65 g{circumflexover (=)}3.0 equiv) at such a rate that the reaction temperature wasmaintained at between −5° C. and 0° C. (about 30 mins). The soliddissolved after the base was added to afford a slightly turbid solution.Stirring was continued for another 2 hrs at below −5° C. to 0° C. After3 hrs the turbid reaction mixture was added slowly, with good stirring,into ice/water (700 ml{circumflex over (=)}35 V) at 5° C. whereby anyundissolved particles were filtered under vacuum. While stirring, the pHwas adjusted to 4.2 using 2M HCl (≈40 ml{circumflex over (=)}≈2 V). Thethick white precipitate was collected by filtration and the damp cakewashed with 200 ml{circumflex over (=)}10 V of water, air dried undervacuum briefly, slurried in 400 ml{circumflex over (=)}20 V of methanoland then stirred under reflux. The suspension was filtered and the dampcake washed again with 200 ml 10 V of methanol then dried under vacuum.The wet cake was then taken in methanol (400 ml{circumflex over (=)}20V) and acetonitrile (200 ml{circumflex over (=)}10 V) and stirred underreflux. The suspension was filtered hot and the damp cake washed with200 ml{circumflex over (=)}10 V of methanol then dried under vacuum. Theproduct was dried under vacuum at 35° C. to a constant weight to affordthe tetrapeptide acid Ac-(AA₁-AA₄).

Output: 120 g; Yield: 62.0%; Purity: 97.1%.

CDEG-1:

CLEU-15 (4.3 g), CBBC-3A (2.24 g{circumflex over (=)}1.0 equiv), andHOAt (0.56 g{circumflex over (=)}1.2 equiv) were charged into an RBFcontaining DMF (˜2.6 ml{circumflex over (=)}6 V). The mixture wasstirred for 15-30 minutes to yield a clear solution at 25° C. and thentreated with DIPEA (1.71 g{circumflex over (=)}4.0 equiv) The reactionwas then cooled to −15° C. and HATU (1.57 g{circumflex over (=)}1.25equiv) was added to the mixture. The reaction mixture was stirred for 2hrs at −10° C., warmed to 20° C. and then stirred for 1 hr at the sametemperature. The reaction mass was added to 10% citric acid solution(270 ml{circumflex over (=)}60 V) and stirred for 30 minutes at 10° C.The precipitated solid was filtered, washed with water (270ml{circumflex over (=)}30 V), 5% NaHCO₃ solution (130 m{circumflex over(=)}30 V) and again with water (270 ml{circumflex over (=)}60 V). Thesolid was dried under vacuum at 35° C.

Output: 4.0 g; Yield: 65.1%; Purity 79.25%.

CDEG:

20% piperidine in DMF (25 ml{circumflex over (=)}5 V) was charged intoan RBF under a nitrogen atmosphere. The solution was cooled to −5° C.and CDEG-1 (5.0 g) was added. The reaction mixture was stirred for 45minutes at 0° C. The reaction mixture was poured into DIPE (250ml{circumflex over (=)}50 V) and then stirred for 15 minutes. Theprecipitated solid was filtered off under nitrogen and washed with DIPE(50 ml{circumflex over (=)}10 V). The solid was then taken up in ethylacetate (125 ml{circumflex over (=)}25 V) and stirred for 1 hr at 25° C.The fine solid obtained was filtered, washed with ethyl acetate (50ml{circumflex over (=)}10 V), and then dried under vacuum.

Output: 4.7 g; Yield: Quantitative, Purity: 87.6%, D-Ser impurity 1.5%,hydantoin impurity 0.16%.

HPLC Condition for CDEG:

Column: YMC basic (250 mm×3.0 mm), 5μ

Mobile phase A: 0.1% TFA in ACN

Mobile phase B: 0.1% TFA in H₂O

Wave length: 226 nm, Diluent: Mobile phase A: M.P.B=27:73

Column tem: 50° C., inject.vol. 50 μl

Gradient T/% A=0/73, 18/70, 41/30, 43/73, 50/73

Flow rate 0.5 ml/min

1. A liquid-phase process for preparing Degarelix having the formulaAc-AA₁-AA₁₀-NH₂:

or a pharmaceutically acceptable salt or solvate thereof; comprising thestep of cleaving an ε-amino protecting group Pε from a Degarelixprecursor according to formula (P₄)(Pε)Ac-AA₁-AA₁₀-NH₂ in an organicsolvent, wherein the organic solvent further comprises a cleaving agentdissolved therein, and the (P₄)(Pε)Ac-AA₁-AA₁₀-NH₂ has the formula:

wherein P4 is a hydroxyl-protecting group or hydrogen.
 2. The processaccording to claim 1, wherein the cleaving agent is chosen fromtrifluoroacetic acid and piperidine.
 3. A liquid-phase process forpreparing a protected Degarelix precursor having the formula(P₄)(Pε)Ac-AA₁-AA₁₀-NH₂:

or a pharmaceutically acceptable salt or solvate thereof, comprising thestep of coupling (P₄)Ac-AA₁-AA₄ with a (Pε)AA₅-AA₁₀NH₂ peptide orcoupling Ac-AA₁-AA₃ with a (P₄)(Pε)AA₄-AA₁₀NH₂ peptide in an organicsolvent, wherein the organic solvent further comprises a peptidecoupling reagent and an organic amine base dissolved therein, wherein Pεis an ε-amino protecting group and P4 is a hydroxyl protecting group orhydrogen, and wherein the peptides have the following formulae:


4. The process according to claim 1, wherein Pε is chosen from oft-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc) andallyloxycarbonyl (Alloc).
 5. The process according to claim 1, whereinthe Pε protecting group is Fmoc.
 6. The process according to claim 1,wherein the organic solvent is DMF.
 7. The process according to claim 3,wherein the peptide coupling reagent is chosen from at least one ofo-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),o-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) and o-(benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlroide (EDC.HCl),(2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium)hexafluorophosphate(HCTU), 2-(benzotriazol-1-yl)oxy-1,3-dimethylimidazolidiniumhexyluorophosphate (BOP), and diisopropylcarbodiimide (DIC).
 8. Theprocess according to claim 3, wherein the organic amine base is chosenfrom at least one of N,N′-diisopropyl ethyl amine (DIPEA),N-methylmorpholine (NMM), triethyl amine (TEA) and2,4,6-trimethylpyridine.
 9. The process according to claim 3, whereinthe solution further comprises a coupling additive chosen from3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt),1-hydroxy-7-aza-benzotriazole (HOAt) and 1-hydroxybenzotriazole (HOBt)dissolved therein.
 10. The process according to claim 3, wherein theorganic amine base is DIPEA and the peptide coupling reagent is HATU.11. The process according to claim 9, wherein the organic amine base isDIPEA and the peptide coupling additive is HOAt.
 12. The processaccording to claim 9, wherein the peptide coupling reagent is HATU andthe peptide coupling additive is HOAt.
 13. The process according toclaim 9, wherein the organic amine base is DIPEA, the peptide couplingreagent is HATU and the peptide coupling additive is HOAt.
 14. Theprocess according to claim 3, wherein the organic amine base is used inan amount ranging from about 2.5 to about 3.5 molar equivalents ofAA₅-AA₁₀ hexapeptide.
 15. The process according to claim 3, wherein theorganic solvent is cooled to a temperature of −10° C. or lower and thereaction is then performed at that temperature.
 16. The processaccording to claim 9, wherein the peptides and coupling additive arefirst dissolved in the organic solvent before adding the couplingreagent and the organic amine.
 17. A liquid-phase process for preparinga Degarelix intermediate having the formula (P₄)Ac-AA₁-AA₄:

or a pharmaceutically acceptable salt or solvate thereof, comprising thestep of hydrolyzing a compound having the formula (P₄)Ac-AA₁-AA₄-R withan alkaline hydroxide, wherein R is a carboxyl protecting group, and P₄is hydrogen or a hydroxyl protecting group, wherein the compound(P₄)Ac-AA₁-AA₄-R has the formula:


18. A liquid-phase process for preparing a hexapeptide (Pε)AA₅-AA₁₀NH₂comprising: coupling of (Pε)AA₆-AA₁₀NH₂ and (P_(X))AA₅, wherein P_(X) isan amino protecting group, AA₅ is 4Aph(L-Hor), AA₆ is D-Aph(Cbm), AA₇ isLeu, AA₈ is Lys(iPr), AA₉ is Pro, AA₁₀ in is D-Ala, and Pε is an ε-aminoprotecting group, to provide (P_(X))(Pε)AA₅-AA₁₀NH₂, and cleaving Pxwith TFA to provide (Pε)AA₅-AA₁₀NH₂, wherein (P_(X))(Pε)AA₅-AA₁₀NH₂,(Pε)AA₆-AA₁₀NH₂ and (P_(X))AA₅ have the following structures:


19. (canceled)
 20. The process according to claim 17, wherein thecompound having the formula Ac-AA₁-AA₄-R is first prepared by couplingAc-AA₁-AA₃ with (P₄)AA₄-R or coupling Ac-AA₁-AA₂ with (P₄)AA₃-AA₄-R, thepeptides having the following formulae:


21. The process according to claim 17, wherein R is methyl or benzyl.22. The process according to claim 17, wherein the alkaline hydroxide isLiON.
 23. Intermediate polypeptides according to the formulae:

wherein R is a carboxyl protecting group, Pε is an amino protectinggroup, and P4 is hydrogen or a hydroxyl protecting group.
 24. A processfor the purification of Degarelix which comprises a purification step bypreparative HPLC using a PLRP—S column.
 25. A solid-phase process forpreparing a Degarelix intermediate having the formula (P4)Ac-AA₁-AA₄:

or a pharmaceutically acceptable salt or solvate thereof, comprising thesteps: a) reacting (PN)AA₂ with

to provide

b) removal of PN from

to provide

c) reacting (PN)AA1 with

to provide

d) if PN is not acetyl, removal of PN from

to provide

and subsequently acetylating

to provide

and e) cleaving

to provide (P4)Ac-AA₁-AA₄, wherein P4 is H or a hydroxyl protectinggroup on AA₄, and PN is an amino protecting group.
 26. A liquid-phaseprocess for preparing a hexapeptide (Pε)AA₅-AA₁₀NH₂ comprising: coupling(P5)AA₅-AA₇ with (Pε)AA₈-AA₁₀NH₂ to provide (P5, Pε)AA₅-AA₁₀NH₂, andsubsequently cleaving P5 to provide (Pε)AA₅-AA₁₀NH₂, wherein P5 is anamino-protecting group on AA₅ and Pε is a side chain amino protectinggroup on AA₆ and wherein AA₅ is 4Aph(L-Hor), AA₅ is D-Aph(Cbm), AA7 isLeu, AA8 is Lys(iPr), AA₉ is Pro, and AA₁₀ is D-Ala.
 27. Theliquid-phase process of claim 26, followed by coupling (P4)Ac-AA₁-AA₄ to(Pε)AA₅-AA₁₀NH₂.
 28. The process according to claim 3, wherein the Pεprotecting group is chosen from t-butoxycarbonyl (Boc),9-fluorenylmethyloxycarbonyl (Fmoc) and allyloxycarbonyl (Alloc). 29.The process according to claim 28, wherein the Pε protecting group isFmoc.
 30. The process according to claim 3, wherein the organic solventis DMF.
 31. The process according to claim 17, wherein the carboxylprotecting group is chosen from a C₁-C₄ alkyl and benzyl group.
 32. Thepolypeptide according to claim 23, wherein the carboxyl protecting groupis chosen from a C₁-C₄ alkyl and benzyl group.